Hashed PBMCs loaded at 16k cells RNA Library: Sample Pool-16
PBMCs were sorted by FACS into naïve T-cells, memory T-cells, and a non-T-cell bulk population. BCells were incubated with TruStain FcX for 10 minutes on ice, followed by staining with antibodies for 20 minutes on ice. Cells were washed with AIM V medium plus 25mM HEPES and sorted on a BD FACSAria Fusion. FACS sorted cells were stained according to the New York Genome Center Technology Innovation Lab protocol (v2019-02-13). One million cells of each population were resuspended in 100 μl of staining buffer: DPBS without calcium and magnesium supplemented with 2% w/v BSA. 10 μl TruStain FcX was added and cells were incubated on ice for 10 minutes, after which they were stained with 0.5 μg of a TotalSeq-A hashing antibody on ice for 30 minutes. Stained cells were washed three times with 1 mL of PBS+BSA. Cells from each population were pooled together in equal numbers, and passed through a 35 μm Falcon Cell Strainer. Libraries were prepared using the Chromium Single Cell 3' v3 reagent kit (10x Genomics, 1000075) following the 10x Genomics User Guide (CG000183 Rev A), with the only modification being cell overloading from 16,000 to 80,000 cells per well. All libraries were sequenced on a NovaSeq S4 flowcell.